Biotin, or vitamin H, is an indispensable element in intermediary metabolism in many organisms since it is an essential factor of biotin-dependent carboxylases important in fatty acid synthesis, gluconeogenesis, and amino acid metabolism. Biotin is useful as a food supplement, a cosmetic additive, and a diagnostic reagent in biotin-avidin-based detection assays.
Most biotin for commercial use is currently produced by a complex chemical synthesis process. Although several investigators are attempting to synthesize biotin in commercial quantities using microbiological methods, the cost thus far has been prohibitive.
The biotin biosynthetic pathway converts pimelyl-CoA to biotin through a series of enzyme reactions. In microorganisms, such as Escherichia coli and Bacillus, most of these enzymes are encoded by genes contained on biotin gene clusters, or operons. Regulation of expression of these genes significantly influences biotin production. Wild type microorganisms, for example, produce only small amounts of the vitamin apparently because such microorganisms exert tight control over expression of the enzymes involved in biotin biosynthesis. Researchers have fed microorganisms pimelic acid and other biotin precursors in order to try to improve biotin production (see, for example, Ogata, pp. 390-394, 1970, Methods in Enzymology, vol. 17a; Izumi et al., pp. 231-256, in Biotechnology of Vitamins, Pigments, and Growth Factors, Elsevier Applied Science, E. J. Vandamme, ed.; U.S. Pat. No. 3,393,129, by Shibata et al., issued Jul. 16, 1968; and U.S. Pat. No. 4,563,426 by Yamada et al., issued Jan. 7, 1986). In addition, a variety of recombinant DNA techniques have been employed to try to improve microbial biotin production including (a) transformation of genes encoding enzymes involved in the biotin biosynthetic pathway (i.e., biotin biosynthetic pathway genes) into microorganisms (see, for example, GB Publication No. 2,216,530, by Pearson et al., published Oct. 11, 1989; U.S. Pat. No. 5,110,731, by Fisher, issued May 5, 1992); (b) feeding of biotin precursors, such as pimelic acid, to such transformed microorganisms (see, for example, European Patent Publication No. 375,525, by Gloeckler et al., published Jun 27, 1990; Sabatie et al., pp. 29-50, 1991, Journal of Biotechnology, vol. 20; Ohsawa et al., pp. 39-48, 1989, Gene, vol. 80; and Ohsawa et al., pp. 121-124, 1992, J. Ferment. Bioeng., vol. 73); and (c) isolation of derepressed mutants for biotin biosynthesis (see, for example Japanese Patent Publication No. 62,155,081, assigned to Shiseido KK, published Jul. 10, 1987; Japanese Patent Publication No. 61,202,686, assigned to Shiseido KK, published Sep. 8, 1986; and Japanese Patent Publication No. 61,149,091, assigned to Nippon Soda KK, published Jul. 7, 1986). These attempts have resulted in some increased biotin production; however, in each case, the amount of biotin produced using such methods is substantially lower than that required for a commercially viable process.
While it is known that pimelyl-CoA is a precursor of biotin and that feeding of pimelic acid to microorganisms can improve biotin production, the intracellular source of pimelic acid and its derivatives (e.g., pimelyl-CoA) was not recognized until the present invention. Several investigators have speculated about the source of pimelic acid. For example, Ohsugi et al., pp. 343-352, 1988, J. Nutr. Vitaminol., vol. 34, and pp. 253-263, 1985, J. Nutr. Vitaminol., vol. 31, discussed the possibility that pimelic acid might be formed from long chain fatty acids, such as oleic, linoleic, and linolenic acids, via a degradation process. Eisenberg, pp. 544-550, 1987, Escherichia coli and Salmonella typhimurium Cellular and Molecular Biology (Neidhardt, F. C. et al., eds., American Society of Microbiology, Washington, D.C.) speculated that pimelyl-CoA might be a product derived from fatty acid synthesis and degradation steps, based on an isotopic study conducted by Lezius et al. (pp. 510-525, 1963, Biochem. Z., vol. 336) in Achromobacter, a study which also led Lezius et al. to conclude incorrectly that biotin production involves a condensation reaction between pimelyl-CoA and cysteine. Eisenberg further speculated that, even if pimelyl-CoA were to be produced using the fatty acid pathway, a mechanism would be required to either add or subtract a carbon from a fatty acid chain (which has an even number of carbons) to form pimelate (which has seven carbons). Eisenberg also suggested that biotin and lipoic acid are very similar and that the process to produce them probably involves octanoic acid as an intermediate. Since octanoic acid has eight carbons, such a process would require removal of a carbon atom to form pimelate. Eisenberg further suggests that the octanoic acid may be produced de novo or by degradation of long chain fatty acids.
The fatty acid biosynthetic pathway converts acetyl coenzyme A (acetyl-CoA) to short-chain and long-chain fatty acids through a series of enzymatic reactions (see, for example, the following review articles: Vanden Boom et al., pp. 317-343, 1989, Annu. Rev. Microbiol., vol. 43; McCarthy et al., pp. 60-63, 1984, Trends Biochem. Sci.; Stumpf et al., pp. 173-176, 1981, Trends Biochem. Sci.; Vagelos , pp 100-140, 1974, "MTP International Review of Science, Biochemistry of Lipid", T. W. Goodwin, ed., Butterworth, London). Although fatty acid synthesis is essentially ubiquitous throughout nature and the general mechanism (i.e., reactions) by which fatty acids are synthesized is similar, the structural form of the enzymes involved in the pathway differs significantly between organisms. The different structures are grouped into two types, which are called Type I fatty acid synthesis (FAS) and Type II FAS. Plants and most bacteria exhibit Type II FAS in which essentially each reaction along the pathway is carried out by a separate enzyme. In contrast, other organisms (e.g., animals and fungi) exhibit Type I FAS in which the reactions are typically carried out by multifunctional proteins that exhibit more than one enzymatic activity. Substantial sequence and functional homologies can be found between the discrete Type II FAS enzymes and corresponding regions on Type I FAS multifunctional proteins.
FIGS. 1 and 2 illustrate the fatty acid synthesis pathway as it is currently known in Escherichia coli. Each of the enzymes involved in this pathway has been identified and the current belief of how the enzymes effect synthesis of fatty acids follows: The first step of fatty acid biosynthesis is the conversion of acetyl-CoA to malonyl coenzyme A (malonyl-CoA). This reaction is catalyzed by the enzyme complex acetyl-CoA carboxylase (ACC) which includes 4 dissociable subunits: biotin carboxylase (BC), which is encoded by the accC gene; biotin carboxyl-carrier protein (BCCP), which is encoded by the accB gene (formerly known as the fabE gene); and carboxyl transferase (CT), which consists of two heterologous subunits encoded by the accA and accD genes. Biotin is an essential co-factor of acetyl-CoA carboxylase.
Malonyl-CoA is then converted to malonyl-acyl carrier protein (malonyl-ACP) by malonyl coenzyme A-acyl carrier protein transacylase, which is encoded by the fabD gene. The acyl carrier protein (ACP) is encoded by the acpP gene. A 3-ketoacyl-acyl carrier protein synthetase subsequently combines malonyl-ACP with either acetyl-ACP or acetyl-CoA (depending on the enzyme) to form acetoacetyl-ACP and carbon dioxide. At least two enzymes can perform this condensation step in Escherichia coli. 3-ketoacyl-acyl carrier protein synthetase III (KAS III), encoded by the fabH gene, can combine acetyl-CoA with malonyl-ACP to form acetoacetyl-ACP. KAS III also has an acetyl-CoA:ACP transacylase activity. In contrast, 3-ketoacyl-acyl carrier protein synthetase I (KAS I), encoded by the fabB gene, can combine acetyl-ACP and malonyl-ACP to form acetoacetyl-ACP. KAS I also exhibits a decarboxylase activity capable of converting malonyl-ACP to acetyl-ACP.
Acetoacetyl-ACP is further elongated, two carbons at a time, by a series of reactions in which malonyl-ACP is added to the chain and carbon dioxide is removed. After each condensation step, the growing fatty acid is reduced by 3-ketoacyl ACP reductase, dehydrated by 3-hydroxyacyl ACP dehydratase, and reduced by enoyl ACP reductase. Another step in the production of unsaturated fatty acids is mediated by 3-hydroxydecanoyl thioester dehydratase (encoded by the fabA gene). This enzyme can convert 3-hydroxydecanoyl-ACP to 2-decanoyl-ACP which the enzyme can then isomerize to form 3-decanoyl-ACP. Yet another enzyme, 3-ketoacyl-ACP synthetase II (encoded by the fabF gene) converts palmitoleoyl-ACP to cis-vaccenoyl-ACP.
Several of the genes encoding enzymes of the fatty acid synthesis pathway have recently been isolated from Escherichia coli. These genes include accA (see, for example, Li et al., pp. 16841-16847, 1992, J. Biol. Chem, vol. 267); accB (see, for example, Li et al., pp. 855-863, 1992, J. Biol. Chem, vol. 267); accC (see, for example, Li et al., pp. 855-863, 1992, J. Biol. Chem, vol. 267); accD (see, for example, Li et al., pp. 5755-5757, 1992, J. Bacteriol., vol. 174); fabA (see, for example, Cronan et al., pp. 4641-4646, 1988, J. Biol. Chem., vol. 263); fabB (see, for example, de Mendoza et al., pp. 2098-2101, 1983, J. Biol. Chem., vol. 258); fabD (see, for example, Magnuson et al., pp. 262-266, 1992, FEBS Letters, vol. 299; and Verwoert et al., pp. 2851-2857, 1992, J. Bacteriol, vol. 174); fabG (see, for example, Rawlings et al., 5751-5754, 1992 , J. Biol. Chem., vol. 267); fabH (see, for example, Tsay et al., pp. 6807-6814, 1992, J. Biol. Chem., vol. 267); and acpP (see, for example, Rawlings et al., ibid.). The accB and accC genes are located adjacent to each other on the Escherichia coli chromosome and are co-transcribed (see, for example, Li et al., pp. 855-863, 1992, J. Biol. Chem, vol. 267). The fabD, fabF, fabG, fabH, and acpP genes are also clustered on the Escherichia coli chromosome (see, for example, Rawlings et al., ibid.; Tsay et al., ibid.).
Two antibiotics, thiolactomycin and cerulenin, have been used to distinguish the enzymatic activities of 3-ketoacyl-ACP synthetases I, II, and III. Thiolactomycin (TLM) selectively inhibits 3-ketoacyl-ACP synthetase I, II and III, possibly by blocking the malonyl-ACP binding sites on the enzymes. Cerulenin is known to inhibit 3-ketoacyl-ACP synthetases I and II. Amplification of the fabB gene, encoding 3-ketoacyl-ACP synthetase I, in an Escherichia coli microorganism, confers TLM resistance to the microorganism (see, for example, Tsay et al., pp. 508-513, 1992, J. Bacteriol., vol. 174).
Attempts to analyze the enzyme components of the fatty acid synthesis pathway have included producing null and temperature sensitive mutants to be used in a variety of growth studies. Two null mutants have been isolated, namely fabA.sup.- and fabF.sup.- mutants. A series of temperature sensitive mutants have been isolated including fabD.sup.ts, fabE.sup.ts (i.e., accB.sup.ts), fabA.sup.ts, fabB.sup.ts, and fabF.sup.ts fabB.sup.ts.
Efforts to increase biotin production by genetic manipulation of components of the biotin biosynthetic pathway have been hampered by a lack of understanding of how carbon flows into the biotin biosynthetic pathway. Increasing pimelic acid by exogenous supplementation of the growth medium has resulted in at least some increased biotin production. There remains, however, a need to improve cellular biotin production by increasing endogenous sources of biotin precursors, for example, by engineering cells to bring more carbon into the biotin biosynthetic pathway in order to overproduce biotin precursors such as pimelyl-CoA.